SU620215A3 - Method of obtaining antibiotic complex possessing antimycotic activity - Google Patents

Description

(54) METHOD FOR OBTAINING ANTIBIOTIC

Of a complex possessing anti-inflammatory activity /

FIELD OF THE INVENTION The invention relates to the microbiological industry, namely the production of antibiotics.

The proposed antibiotics are new to the patent and science “technical literature are not described.

The proposed method for producing an antibiotic complex with antifungal activity is that the Asperg-ieeusrugviosu strain HRR 8113 is grown under aerobic conditions on a nutrient medium containing nick carbon, nitrogen and mineral solv, followed by release of the target product and its separation into components .

The AspergrietOb rogXjCosue NRftt48113 strain was isolated from soil taken from the ruins of Pompeii, Italy, and is stored in the crop collection of the research laboratory of the northern US Department of Agriculture, Perry, Illinois 61604, numbered NRRU 8113, and Harak. It is characterized by the following properties

Agar čapek. Culture restet slowly, reaching 1.5-2.0 cm in diameter for 15 days at. 25 ° C. The surface of the colony is convex and velvety, it wrinkles with time in the center and then cTaHOi. The convex periphery of the mycelium is a strip 2 mm wide, deeply submerged, colorless, and is curved. Brownish exudate is formed at the ends of air hyphae. Forms a pale purple soluble pigment.

After 5 days, the surface of the colony acquires a color from white to yellow-brown of brown color, the reverse side of the colony is brown-orange in the center and brownish to brown-purple at the periphery. After 1–2 days, the colonies become a yellowish pink. After 14 days, the colonies become light gray-red, the terminal surface becomes warty and bright yellow. At the top and at the ends of the colony, randomly spread dark yellow tufts of cell membranes appear. With time) 2

the yellow plaques and the end surface become yellow-green. After 3 weeks orange-purple gon is observed on new surface end components. First, the reverse side of the colony is slightly concave. As the aging process takes place, the holon becomes flat to the surface of the agar and the back side slightly softens. After 10 days, the reverse side becomes light brown, and after 15 days, it becomes grayish-red.

The face of the raebrossa on the surface is sometimes slate or in the field. submersible strip. Heads of conidia, first radiant and spherical; over time, they can develop into short bars that are more compact. Spherical heads with a diameter of microns average 50 microns. Columnar heads have a length of up to 140 microns, a width of 70 microns

Conidia are spherical to hemispherical, small-folded, greenish-gold in mass. Their diameter is 2.8-3.9 microns, an average of 3.2 microns. Colorless sterigma / 1 of the primary sterigm is 4.7–11.0 µm, an average of 7.9 µm, E has the widest point of 2.4 µm, and narrows down to 1.6 µm.

Secondary sterigms may occur as single or doubles, they are derived from primary ones and have a flat shape. In the widest place, the sizes are 3.0 µm and narrow to 0 µm, where they become tubular. The tubular tip has a length of 1.2 microns. The total length is 5.5–12.6 µm, in cpefl-i it is 9.2 µm.

The vesicles are spherical to hemispheres of Cesca, they can be apically flat, becoming brown with time. Their diameter is 7.4-11.2 microns; on average 9.4 microns.

Endophores are smooth, relatively thick-walled, at first they are hyalinoyl, aateM turns into light brown. They are slightly wider in the vesicle and can collapse on a cone near the stacks of cells. The average width of 5.9 microns. Kottshdisporyy have a length of 47.7-166.6 microns, in the middle of 10.0 microns. They arise from submersible gvEf or horizontally from air

gffovy threads.

Askogwd condition is observed after 20 days. A large yellowish bundle of cell membranes, arising on the surface, can be found on any mipeda. They consist of cell membranes within which there is an ossh or several gelatinia. The cell wall is spherical, semi-tular.

154

oval or elongated, they are thick Hfjie and hyalite. The diameter of the globular membranes of the cells is 18-24 µm, an average of 21.8 µm.

Cleistothecia spherical to hemispherical, thick-walled (cf.) abnormally viscous and fibrous. At first they are relatively colorless, they become red-purple and darken with time.

Their diameter is 165-470 microns, an average of 275 microns.

Malt agar. The colonies grow at 25 ° C, expand rapidly, reaching 4-5 cm in days. First, the gray-white colonies become moderately olive-green after 4 days. Small yellowish beams: the cells of the enveloped cells are dotted along the edge and randomly scattered around the ferro-like surface of the agar. After 20 days, these bundles of cell membranes cover a large part of the surface. The reverse side of the colony is greyish-yellow. After 15 days, an ascogenic state is formed. Small yellowish bundles of cell membranes that form on the surface can be found at any level in the mycelium. They consist not of cell membranes, inside of which there is one or more gluestothecia. Cell membranes may cover large areas above conidia heads. Cell shells are spherical or hemispherical, oval or elongated, thick-walled and hyaline. The diameter of the globular frills of the cells is 18-24 µm, an average of 21.8 µm.

Cleistothecia spherical or hemispherical, they are painted in dark red brown color. The diameter of 389-700 microns with an average of 506 microns.

Leki are fragile, hyaline, hemispherical to oval. The diameter of the hemispheric asc is 8.7-11.9 µm, on average, Yu, 3 µm. The size of the oval convicts 10, 2x8.7 - 10.3 microns, an average of 12.2X9.1 MKMi

Ascospores are red-orange, small-folded, with two parallel products in one equipolar comb, with a width of up to 0.8 microns. i Ascospores looked two-convex along the longitudinal axis. If the scallop is peripheral, then the ascospores are spherical. The diameter of the ball 4.9-6.3 microns, an average of 5.4 microns.

This strain can vyrashivat on nutrient media containing carbon notochniki, n example glucose, molasses, starch, lactose, sucrose, maltose, glycerin, aayuta SOURCES, nairvymer, casein hydrolyzate ekzimatichesky ne-pton, glutamate, mineral salts and mikroeleme-Options. For producing significant amounts of the mixture, antibiotic A-30912 prefer to submerge the aerobic enzyme in the fermenters. Small amounts of anthinyogic A 30912 can be shaken in a culture. Following a large time interval in the production of an antibiotic, usually associated with the inoculation of large plants with spore forms of microorganisms, it is preferable to use a vegetative inoculum. Vegetative monk mind. Prepare by inoculating a small volume of culture with spore forms or mycelial cells of the microorganism and obtain an actively growing culture of the microorganism. The vegetative inoculum is then transferred to the reservoir. The medium used to grow the vegetative inoculum may be the same as for large fermentation, but other media may be used. Microorganism growing antibiotic A-309.12 can be grown at 2 ° -43 ° C, micro organism propagates well at 25 ° C. Right production of antibiotic A-30912 occurs at 23 ° C. Sterile air is blown through the fermentation medium. For the affective production of the antibiotic, the volume of air produced in the tank is about 0.4 volume of air per volume of culture medium per minute. The production of antibiotic A 30912 is controlled during the fermentation by sampling alcohol from extracts of the culture liquid for antibiotic action against the microorganism sensitive to the antibiotic A-ZO912, for example, Candida abstape. Bioanalysis is carried out using paper discs on agar plates. As a rule, antibiotic activity is detected on the second day of fermentation. Maximum production of antibiotic activity is usually achieved between the third and sixth days. Example, Preparing an Aspet gi e .U5 rugu o5UfiNRRb 8113 culture and storing it on 18U150 ml beveled agar of the desired composition,%: Dextrin1, OOOO Enzymatic Hydrolyzate breech .2000 o.yooo Yeast extract 0.1000 Extract of HNP 0,02 M-ZO-THNZO 0.0200 0.0004 98.5596 The inoculated vegetation medium is incubated in a 25 O ml wide-neck Erlenmeyer flask at 25 G for 24 hours on a rocking chair (250 rpm). This incubated vegetation medium can be used directly to inoculate the vegetation medium from the second stage. It is better to store it for later use by preserving the culture in the vapor phase of liquid nitrogen. The culture is prepared for such storage in several small loops as follows; 2 ml of incubated vegetation medium and 2 ml of glycerin-lactose solution of the following composition are placed in each pot;%: Glycerol-2O Lactose-1O Deionized water 7O The resulting suspensions are stored in the vapor phase of liquid nitrogen. The suspension (l ml) is used to inoculate 5 O ml of fermentation medium from the first stage, the composition of which is the same as for the described fermentation medium / Inoculated medium from the first stage is incubated in a 250 ml wide-mouth Erlenmweyer flask at 25 ° C for 22 hours on the rocking chair (25O r / min). To obtain a larger volume, 10 ml of the described first-stage fermentation medium are used to inoculate 4OO ml of the second-stage fermentation medium of the same composition as the first-stage medium. The second-stage sressa is incubated in a 2-liter Erlenmeyer flask at 25 L for 25 hours on a rocking chair (250 rpm). The incubated second stage fermentation (8OO ml) / prepared, as mentioned above, is used to inoculate 1OO l of sterile fermentation medium of the following coctaaa, g / l: Glucose25. Starch10 Peptone.1O Black treacle5 Casein-4 enzymatic hydrolytic

 , 0

xe0.5

ShASO, 2, O

FeSQ lHjO,

(dissolved in.

2 ml of concentrated it) O, O04

Deionization

water up to, l1,0,

After sterilvaapnn in an autoclave at 121 C for 3.0 min at a pressure of 1 atm., 1 atm. PH of the medium 6.8. The inoculated medium is fermented in an enzyme torus with a capacity of 1–5 l at 25 ° C for 4 days. The enzyme paravail medium is flushed with sterile air at a flow rate of 0.5 volume / volume / min. The fermentation medium is stirred with a conventional stirrer at 3oo rpm.

2OO l of the culture liquid is thoroughly mixed with 200 p of methanol for 1 hour, then filtered on a filter from diatomaceous earth. The pH of the filter is adjusted to 4 with the aid of 5N.N. The acidified filtrate is extracted twice with equal volumes of chloroform. The chloroformate extracts are combined and evaporated in vacuo to a volume of about 4 liters. This concentrate is added to 60 L of diethyl ether to precipitate a mixture of A-3X912. The precipitate was separated by filtration, dried, and 38 g of antibiotic A-330912 were obtained in the form of a gray powder. The filtrate is evaporated in vacuo to give an oil; this oil is dissolved in 50 ml of methanol. The methanol solution is added to diethyl ether (7.5 L) to precipitate an additional amount of antibiotic A-3X912. The precipitate is also separated by filtration, dried, and 3.5 g of antibiotic A-30912 are obtained.

PRI mme R 2. Selection of the component A of the antibiotic A-ZO912.

Antibiotic A-30912 complex (20 g was introduced into a silica gel column (4X107 s in acetonitrile-water (95: 5). The column was eluted with acetonitrile-water (95: 5) at a flow rate of 1-2 ml / min / fraction was collected 2 мл ml. These fractions. &Amp; 1CHN are checked by thin-layer silica gel chromatography using a mixture of acetoniouryl-water (95: 5) and Candidcuae bicc ns biologic. Fractions 74 to 125 are combined and evaporated. The concentrated solution is crystallized and 124 are obtained. mg sterigmatocystin. Fractions with

212 to 273 are combined and evaporated at B under vacuum to give an oil. This maolo is dissolved in a small volume of methanol. The methanol solution is added to

diethyl ether (l5 volumes). The precipitate is separated and dried to obtain 23 mg of component D of antibiotic A-30912. Fractions 274 through 473 contain components A, B C, and D of the antibiotic A-30912. Fractions 482 to 900 contain components A, E, F, and G of antibiotic A-ZO9.12. Fractions 438 through 481 are combined and evaporated in vacuo to give an oil. This oil is dissolved in a small volume of methanol, the methanol solution is added to diethyl ether (15 volumes). The precipitate is separated and dried, yielding 2.17 g of Chonent A cocoon A of antibiotic A-30912.

Partially purified antibiotic A-30912 containing components B, C and D of antibiotic A-30912 (75 O mg) from fractions 274-437 is chromatographed on a gel column (2.2 x 51 cm), fractions of about 1 ml are collected and eluted with the following solvents:

Fractions:

1-25 Acetonitrile

26-62Acetonitrile 1% water

63-700 Acetonitrile + 1.5% water.

Fractions from the column are monitored by thin-gel thin layer chromatography using a mixture of acetonitrile-water (95: 5) and benzene-methanol (7: 3) and Sopdido aeticans biofeedback. fractions 535-685, in which the component D of the antibiotic A-ZO912 is contained, are combined, evaporated in vacuo to do-oil. This oil is dissolved in a small volume of methanol and added to diethyl ether (l5 volumes. The resulting precipitate is separated by filtration, dried, and 69 mg of component D of antibiotic A-30912 are obtained.

Partially purified complex of antibiotic A-ZO912 (18 g), containing components A, B, C, and D A-30912 and fractions 212-437, is dissolved in a minimum volume of acetonitrile-water (4: 1) and chromatographed on a column with an oxide aluminum (s, 8Х56 cm) # collect fractions of about 2 ml. The column was eluted with the following solvents:

Fractions:

1-300 Acetonitrile-water (4: 1)

301-509 Acetonitrile-water (7: 3),

f

Fractions from the column are checked by silica gel thin-layer chromatography, as described above. Based on these data, the fractions are combined and evaporated to aool, the oily residues are dissolved in a small amount of methanol, and methanol pacTBOpj is precipitated with 10-15 volumes of diethyl ether. Connection Method

To obtain purified complex C, a fraction of 6-28 (2 g) is dissolved in methanol, absorbed on a sufficient amount of silica gel, dried and introduced into the upper part of a silica gel column (1.9X80 cm) filled with acetonitrile. The column is eluted with acetonitrile supplied. flow rate of 2 ml / min, collecting fractions of about 10 ml. Per fraction 117, the solvent is changed to a mixture

Component A of antibiotic A-30912, which has antifungal activity, has:

The empirical formula Cy.5, H ,, .. Elemental composition,%:

C 56,52

H7.29

N8.68

about 27.09

Molecular Weight 1100. Cotl -44 ° GS 0.5; CH50N)

id.lll- -ise (with about, 5: SNZOn)

The maximum absorption frequencies in the ultraviolet spectrum (in acidic and neutral methanol) are 225, 275, 284 nm in (H.-LONG methanol 245 and 290 nm.

aetonitrile-water (99: l). Fractions from koponka control thin layer chromatography. Based on the data of thin layer chromatography, the fractions are combined, evaporated to an oily residue, the oily remains are dissolved in a small volume of methanol,

thieves are precipitated with 10-15 volumes of diethyl ether. The content of the components and the weight of the fractions are given in table. 2

Tablida2

Maximum Infrared Absorption Frequencies (KBr):

2.97 (strong), 3.39 (medium), 3, 47 (weak), 5.99: (strong), 6.10 (strong), 6.49 (medium), 6.56 (medium), 6.9 O (medium), 8.00 (weak), 9.13 (weak /, 11.77 (weak) μm.

Maximum frequency of oscillations at a dermal magnetic resonance (in perdimeter methanol):

(176.1, 174.3, 173.4, 172.7 169.8 158.4, 132.8, 130.9, 129.6, 129.0, 116.2, 77.0. 74.4, 75 , 7, 71.3, 70.9,69,6, 68, 4, 68.7, 56.9, 56.1.52.9, 39.0, 38.5, 36.8, 35.2, 33.9, 32.9, 32.6, 30.7, 30.4, 30.2, 2.2 fractions, the weight of the substance obtained and the content of the component in the combined fractions are given in Table 1. Table

27.0, 26.5, 23.6, 20.1 / 19.6, 14, -1 and 11.3 ppm.

The titratable group with hpgPa is 312.8 (initial pH 6.9) in a 6% solution of Dimethylformamide.

A white amorphous substance soluble in polar organic solvents, for example, methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate is not soluble in nonpolar organic solvents, for example, diethyl and petroleum ethers.

Component D of antibiotic A-30912, which has antifungal activity, has:

ELECTRIC COMPOSITION,%:

C 56.37 H 8.17

N 8.54,:

About 26.92

Molecular Weight.

oLjy 0 ° (C 0.34;).

Mavh: the ultraviolet absorption spectrum (in acidic and neutral methanol) is 225 and 275 nm, mostly 240 n 290 nm.

Maximum absorption frequencies in the infrared spectrum ():

2.98 (strong), 3.31 (weak), 3.3 (shoulder), 3.40 (medium) |;, 3.48 (weak), 5.76 (weak), 6.01 Gsilny), 6.10 (shoulder 6.49 (medium), 8.57 (medium), 6.90. (Medium) 7.81 (last 6) .8.07 (weak), 9.16 (weak).

White amorphous substance soluble in methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, in aqueous solution, especially with a pH greater than 7.0 | insoluble in diethyl oil and petroleum ether.

Component B of antibiotic A 30912 has:

Elementary composition,%: C 57.36 and 5.92

N 8.75

About 26L ..

 -170 (with 0.5 | CHjOH).

   47 (C 0.5; SNZON)

The maximum absorption frequencies in the ultrafluorescence spectrum (sour and neutral methanol) are 223 and 278 nm, in the ooinew 242 and 292 nm.

Maximum absorption frequencies in the infrared spectrum (KBr); 2.99, 3.49 6.09, 6.15, 6D5, 6.54; 6.61, 6.94, 7.62, 8.07, -. 9.26 and 9.39 microns.

Titrated group with a pKoi value of 13.0 (initial pH 7.9 l) in a 66% aqueous solution of dimethylformamide.

A white amorphous substance soluble in methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate and in solutions with a pH above 7.0; insoluble in ethylene and petroleum ethers.

Component C antibiotic. A-ZO912, has;

. Elementary composition,%: C 56.76 H7.88

М 10.61 О 25.09 сЛЗ | “33 (Co, 5; SMEON).

c13 lie (C 0.5 CHgOH)

The maximum absorption frequencies of the ultraviolet spectrum (in acidic and neutral methanol) are 223 and 275 nm, in the base 240 to 290 nm.

The maximum absorption frequencies in the infrared spectrum (KBr) are 2.98, 3, 30.3.43, 3.51, 6.01, 6.12, 6.47, 6.90; 7.04, 7.22, 7 , 38.8.00, 8.30, and 9.13 microns

Titrated group with a pKa value of 13.08 (initial pH 7.9z) in a 66% aqueous solution of dimethylformamide.

White amorphous substance, soluble in methanol, ethanol, dimethylformamide dnmethleesulfoxide, etg-shaacetate, and in aqueous solutions with pH above 7.0; not soluble in diethyl petroleum ether- pax.

Divisions 1B of the components of the antibiotic A 30912 mixture are separated and identified by thin layer chromatography. The best sorbent is silsagel; benzene-methanol (7; 3 pbyem / is the best solvent.

The magnitude of the components of antnbiogika A-30912 with the use of thin layer. chromatography on silica gel with solvent mixtures of benzene methane (7; 3), binnom. The CbtididQkC No. taiecirts shnm mshfooratshzm are listed in Table. 3

Komplekt complex of Ag30912

BUT

In C

d

Components A and D of antibiotic A-ZO912 15 possess antifungal active substance.

This activity is measured with the help of the usual: disk-diffusion technique (6-mm swabs are immersed in solutions containing antibiotics, the swabs are placed on 20

Candida aetoicohs

T icfiopb (ton mentoig-rophsiteft

Component A of antibiotic A-ZO912 against dermatitis

rpiciiophvton mentoi fophsrtes (ticbopb ton g a ctinoie Trictiophvton meg - inihi Tr ic1ioph) ton auinci eanum

Table3

R.

Oh, 35 0.45 0.54 0.59

agar plates, examined by the organism under test). In tab. Tables 4 and 5 show the minimum inhibitory concentration (mic) on the disc, in which antibiotic A-30912 inscribes listed microorganisms.

Table 4

0.5

O, 625 0.07 0.07 8

T a b l and c a 5

1.25-0.039

1.25 0,0195

1.25

 O, OQ98 0,0195 0, O195

1.25-O, 156 O, 156-O, O38